Fastidious Gram-negative Fastidious Gram-negative bacteriabacteria

Bacterial vaginosis, HACEK infections, Legionella

Prof. Cary Engleberg, M.D.Division of Infectious Diseases,Department of Internal MedicineDept. of Microbiology & Immunolog

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Gardenerella vaginalisMobiluncus spp.HACEK (including

Actinobacillus)Legionella spp. (L. pneumophila)

associated with bacterial vaginosis

rare causes of endocarditis;

(juvenile periodontitis)

severe pneumonia(Legionnaires’ disease)

Gram-negative, intracellular pathogens, acquired from the environment

Gram-negative normal flora

Case: vaginitisCase: vaginitis• A 32 year old woman has a vaginal discharge. She has had

no pain, vaginal bleeding, or excessive weight gain. She is G2P2. LMP was two weeks ago and was normal and on time. She is monogamous with her husband.

• On examination, there is a malodorous, light gray discharge at the vaginal introitus.

• Application of a pH strip to vaginal wall =7.0. • Addition of 10% KOH to a sample of the discharge on a slide

produces an intense amine odor (“fishy”). A saline preparation of the discharge shows the following:

“Clue” cells

M Rein, CDC Public Health Image Library, #3720

Clue cell Gram stainClue cell Gram stain

Source undetermined

Questions to considerQuestions to consider• Why is the discharge malodorous? What is

the “fishy” odor?• What is the significance of the vaginal pH?• What is the significance of bacteria-coated

cells (“clue” cells)?• Should her husband be examined and

treated?

Bacterial vaginosis (BV)Bacterial vaginosis (BV)• Overgrowth of vaginal flora with G. vaginalis,

Mobiluncus, Prevotella, Peptostreptococcus, and many other anaerobic species

• Displaces normal lactobacilli (responsible for vaginal acid production; pH<4.5)

• Anaerobic bacteria produce amines which release ammonia in 10% KOH

• Resolves with oral or topical metronidazole

Gardenerella vaginalisGardenerella vaginalis• facultatively anaerobic, nonsporulating,

nonencapsulated, nonmotile, pleomorphic, gram-variable rod

• found in 15% - 69% of women without BV and in 13.5% of girls.

• found in all cases of BV• risk of bacteremia in pregnant women, post-

abortion, and post-hysterectomy

MobiluncusMobiluncus spp. spp.• anaerobic, slowly growing, motile, Gram-

variable, curved bacilli• found in 97% of women with BV but in a

minority of healthy controls• susceptible to most antibiotics, but

resistant to metronidazole – ? role in BV

What is the role of these bacteria in BV?What is the role of these bacteria in BV?• unclear. . .• Koch’s postulates not satisfied by any single agent• Note: BV is not sexually-transmitted disease• When is it necessary to treat?

• HIV-infected• Pregnant• Before GYN surgery• At risk for other STDs

Questions to considerQuestions to consider• Why is the discharge malodorous? What is

the “fishy” odor?• What is the significance of the vaginal pH?• What is the significance of bacteria-coated

cells (“clue” cells)?• Should her husband be examined and

treated?

The The HACEKHACEK group groupH: Haemophilus aphrophilus and H.

paraphrophilusA: Actinobacillus actinomycetemcomitansC: Cardiobacterium hominisE: Eikonella corrodensK: Kingella kingii

Characteristics of HACEKCharacteristics of HACEK• all are Gram-negative, pleomorphic rods• all are normal flora of the human mouth• rare cause of “culture-negative” endocarditis

(particularly after dental work)• fastidious; very slow to grow in culture

(require 5-10% CO2)

• most have beta-lactamase enzymes

ActinobacillusActinobacillus• Found in 20% of adult/teenage mouths• Cause of juvenile and adult periodontitis

– with Porphyromonas gingivalis– In 90% localized, aggressive periodontal

infections with loss of teeth and bone

• Can cause infections mimicking Actinomycosis (usually neck, face, lungs and chest wall) - rare

““Juvenile periodontitis”Juvenile periodontitis”

Erste-zahnartzmeinung.de

Treatment of HACEK organismsTreatment of HACEK organisms

• Endocarditis: ceftriaxone, not ampicillin or penicillin

• Human bites: amoxicillin-clavulanate, fluoroquinolone

• Severe periodontitis: tetracycline

LegionellaLegionella ((primarily primarily L. pneumophilaL. pneumophila))

• Legionnaires’ disease• Pontiac Fever

Case: pneumonia after travelCase: pneumonia after travel• A 55-year-old male automobile dealer was hospitalized

with high fever and cough. • Seven days earlier, he developed symptoms of fever

(38.5C), headache, and generalized muscle ache. The following day, he developed a hacking cough with minimal sputum production.

• He was evaluated 4 days before admission, and a diagnosis of community-acquired pneumonia was made after a chest x-ray which a left lower lobe infiltrate.

Source undetermined

Case (continued)Case (continued)• He was treated with an oral cefuroxime. However, his fever

increased, and he developed watery diarrhea.• His past medical history was unremarkable, but he is a cigarette

smoker. He returned from a 2-week vacation with his wife, three children, 75 year old mother in the Florida Keys (Hawthorne Suites) 2 days before onset. None of his family or co-workers were also ill.

• On admission, temp=39.8oC, heart rate =90/minute. O2 saturation=81% on room air

• WBC=13,700/mm3. • Sputum Gram stain: numerous PMNs, no bacteria.• A chest x-ray showed extension of the right lower lobe infiltrate and

an extensive new left lower and left upper lobe infiltrate.

Source undetermined

Case (conclusion)Case (conclusion)

• Azithromycin was added to the patient’s antibiotic regimen to treat Legionnaires’ disease. This diagnosis was confirmed by a positive Legionella urine antigen test.

• The patient began to improve 48 hours later and recovered. Respiratory secretions obtained from his endotracheal tube grew L. pneumophila SG1 after 3 days.

• A call to the Florida Department of Public Health confirmed that 5 other recent patrons of the Hawthorne Suites had also developed severe pneumonia, and a hot tub at the hotel was positive for L. pneumophila SG1.

Questions to considerQuestions to consider• Was the hot tub the source of the illness? Why?• Why were none of the patient’s family members

or co-workers affected?• Why was the sputum Gram stain negative?• Why was cefuroxime ineffective?• Why was the diagnosis made by a urine antigen

test instead of a culture?

The Belleview-StratfordHotel, Philadelphia

(where the first recognized outbreak of Legionella infection occurred)

Jack E. Boucher, Historic American Buildings Survey

Scope and consequences of the 1976 Scope and consequences of the 1976 Bellevue-Stratford outbreakBellevue-Stratford outbreak

• 182 American Legionnaires become ill• 146 were hospitalized• 29 died (associated with respiratory failure)

• There were no secondary cases

Fibrinopurulent pneumoniaFibrinopurulent pneumonia

Source undetermined

LegionellaLegionella: difficult to distinguish from : difficult to distinguish from the backgroundthe background

Source undetermined

Laboratory investigationLaboratory investigation• Etiologic agent was unknown for months!• Eventually, the infection was passed from

the lung tissues of deceased patients to guinea pigs.

• Then, from guinea pigs to highly-enriched liquid media

• Then, a specialized agar medium

Buffered charcoal yeast extract Buffered charcoal yeast extract agaragar

Supplemented with cysteine and iron pyrophosphateSource undetermined

Outbreaks of legionellosis that preceded the 1976 Outbreaks of legionellosis that preceded the 1976 Philadelphia outbreakPhiladelphia outbreak

Est. Case- attack fatalityLOCATION Year n rate rate

St. Elizabeth’s Hosp., D.C.Health Dept., Pontiac, MichiganJames River, VirginiaBenidorm, SpainOdd Fellow’s Conv., Phila. PA

American Legion Convention, Philadelphia, PA

19651968197319731974

1976

81144 10 89 11

182

1.4% 95%100% -- 2.9% 4.0%

17% 0 0 3.4%10%

17%

Legionnaires’ disease Pontiac fever

• fever, headache, myalgia for ~ 2 days• no other significant clinical signs or abnormal laboratory tests• no fatalities• no pneumonia• incubation period: 24-48 h• L. pneumophila cannot be detected or isolated from patients

• fever, cough, dyspnea, & various other symptoms• GI, hepatic, renal, and neurologic manifestations common• fatal in 5 - 25%• fibrinopurulent pneumonia• incubation period: 2-10 d• L. pneumophila can be isolated from patient samples

Identification and taxonomyIdentification and taxonomy• ~50 Legionella spp. (19 have caused human

disease)• L. pneumophila causes most of human disease

– 16 distinct serogroups– Most disease due to serogroup 1 (SG1) - ~60%

• Features:– All are flagellate GNRs, catalase-positive– Survive major temperature extremes (up to 55oC)– Identified with group-specific antisera

• LLAPs (cultivable only in amoebae)

Aqueous sources of LegionellaeAqueous sources of Legionellae

Biofilms within residential and institutional water systems

liveLegionella

Surface water sourceLegionella + protozoa

Infectious aerosols Microaspiration

Water treatment plant chlorination

Fountains FaucetsShowers

Evaporative cooling towers

Bacterialgrowth inhot water heaters

Evidence that Intracellular Infection is Evidence that Intracellular Infection is Essential for Legionnaires’ DiseaseEssential for Legionnaires’ Disease

• From animal models– Max. growth ~ bacteria associated with cells.– Susceptibility of an animal species ~ susceptibility of its

macrophages to infection in vitro.– Mutants with poor macrophage growth ~ avirulent.

• From human infection– Intracellular bacteria are seen in lung sections.– Antibiotic efficacy ~ penetration of agent into cells.

HOST A HOST B

Human lung is a dead end

for Legionella.

Infection Infection

Transmission

Disease

Aerosol

Protozoa are the natural hosts for Protozoa are the natural hosts for LegionellaLegionella..

Inhalation

Alveolar Macrophages

HOST A HOST B

Legionnaires’ Disease

Infection Infection

Transmission

Disease

Motile,flagellaterods

Association with ER

Delayedphago-

lysosomalfusion

Expressionof flagella & release

Legionella EMsLegionella EMs

Source undetermined

The Dot/Icm ComplexThe Dot/Icm Complex

Effector

macrophagecytoplasm

macrophagemembrane

bacterial OM

bacterial IM

bacterial cytoplasm

periplasm

Legionellakilled by thelysosomes

Dot/icmmutants

Growth-phase regulation of virulenceGrowth-phase regulation of virulence

Optical Density

Time

Exponential Postexponential

“REPLICATIVE” “TRANSMISSIVE”

In v

itro

In v

ivo

Clinical diagnostic methodsClinical diagnostic methods

Culture: sputum (with acid) 20-80% 100%BAL 80-90% 100%lung tissue 90-99% 100%

DFA 25-75% 95-99%

Serology SG1: seroconversion 70-80% 95-99%single titer > 256 10% 50-70%

Urine Ag-RIA (3 to >300 days) 80-98% (SG1) 99%

Sensitivity Specificity

*accurately diagnoses only SG1 = 60% of cases

*

Mortality from Legionnaires’ disease in 3 Mortality from Legionnaires’ disease in 3 outbreaks, by antibiotic therapy*outbreaks, by antibiotic therapy*

Philadelphia Vermont Wadsworth, CA 1976 1977 1978

CephalosporinsAminoglycosidesPenicillinsErythromycin

46%36%23%11%

17%19%16% 4%

25% (80%)§

7% (24%)

* Tsai et al. Ann Intern Med 1979; 90: 509 Broome et al. Ann Intern Med 1979; 90: 573 Kirby et al. Medicine 1980; 59:188

§ parentheses indicate immunosuppressed patients

Uptake of Antibiotics by Pulmonary Alveolar Uptake of Antibiotics by Pulmonary Alveolar MacrophagesMacrophages

taken from Johnson et al. J. Lab Clin Med 1980; 95: 429-39taken from Johnson et al. J. Lab Clin Med 1980; 95: 429-39

C:E ratio ANTIBIOTIC at 120 min

Erythromycin 20.6 ± 3.1Chloramphenicol 2.1 ± 0.2Rifampin 1.8 ± 0.3Tetracycline 0.9 ± 0.1Gentamicin 0.6 ± 0.1Cefazolin 0.07 ± 0.06

Antibiotic rx of Legionnaires’ DiseaseAntibiotic rx of Legionnaires’ Disease

LONGSTANDING CHOICE:

High-dose Erythromycin + RifampinTRADITIONAL ALTERNATIVE:

DoxycyclineBETTER ALTERNATIVES:

Azithromycin and newer macrolidesFluoroquinolones

Questions to considerQuestions to consider• Was the hot tub the source of the illness? Why?• Why were none of the patient’s family members

or co-workers affected?• Why was the sputum Gram stain negative?• Why was cefuroxime ineffective?• Why was the diagnosis made by a urine antigen

test instead of a culture?

What should be done in Ghana?What should be done in Ghana?

• Frequency of this infection is not known• Rapid diagnostic tests unlikely to be available• Treat severe pneumonia on suspicion:

– Patients on steroids or other immunosuppressive medications

– Recent overnight travelers– People with exposure to heated water or water aerosols

• Therapy includes azithromycin, a fluoroquinolone, or erythromycin + rifampin

Additional Source Informationfor more information see: http://open.umich.edu/wiki/CitationPolicy

Slide 6: M. Rein, CDC/Public Health Image Library, Clue cells, #3719, http://phil.cdc.gov/phil

Slide 7: Gram stain of a clue cells, source undetermined

Slide 17: Juvenile Periodontitis, http://www.erste-zahnartzmeinung.de/zahnwissen-kzvwl/zahnwissen/pa_klass.htm

Slide 21: X-ray, source undetermined

Slide 23: X-ray, source undetermined

Slide 26: Jack E. Boucher, “Belleview-Stratford Hotel,” Wikipedia Commons, http://commons.wikipedia.org/wiki/File:BelleviewStratford.jpg

Slide 28: Fibrinopurulent pneumonia, source undetermined

Slide 29: Legionella Gram stain, source undetermined

Slide 31: source undetermined

Slide 40: source undetermined